Name: inducible t7 rna polymerase-mediated multigene expression system, pmgx
Uploaded: 2017-06-27T13:00:00
Description: Watch this Scientific Journal Video about Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX at JoVE.com

Dette arbejde blev støttet af Canadas naturvidenskabelige og tekniske forskningsråd.

1. Opnåelse af interesser <ol><li> Design syntetiske gener. <ol><li> Optimer en gensekvens for E. coli ekspression. </li><li> Fjern eventuelle problematiske restriktionssteder fra sekvensen (AvrII, NdeI, EcoRI og XbaI). </li><li> Indarbejde restriktionssteder for kloning; Et 5&#39;-NdeI site og et 3&#39;-EcoRI site anbefales. Andre steder kan bruges, hvis det er nødvendigt; Henviser til multikloningsområdet for det valgte plasmid ( figur S1- S4</strong...

<ol>
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Commun. 203 (10), 1326-1335 (2016).</li><li>Romier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-expression+of+protein+complexes+in+prokaryotic+and+eukaryotic+hosts:+experimental+procedures,+database+tracking+and+case+studies.">Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies.</a> Acta Crystallogr. D, Biol. Crystallogr. 62 (Pt 10), 1232-1242 (2006).</li><li>Tolia, N. H., Joshua-Tor, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+protein+coexpression+in+Escherichia+coli.">Strategies for protein coexpression in Escherichia coli.</a> Nat. Methods. 3 (1), 55-64 (2006).</li><li>Chanda, P. K., Edris, W. A., Kennedy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+set+of+ligation-independent+expression+vectors+for+co-expression+of+proteins+in+Escherichia+coli.">A set of ligation-independent expression vectors for co-expression of proteins in Escherichia coli.</a> Protein Expr. Purif. 47 (1), 217-224 (2006).</li><li>Kim, K. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-promoter+vector+is+highly+efficient+for+overproduction+of+protein+complexes.">Two-promoter vector is highly efficient for overproduction of protein complexes.</a> Protein Sci. 13 (6), 1698-1703 (2004).</li><li>Tan, S., Kern, R. C., Selleck, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pST44+polycistronic+expression+system+for+producing+protein+complexes+in+Escherichia+coli.">The pST44 polycistronic expression system for producing protein complexes in Escherichia coli.</a> Protein Expr. Purif. 40 (2), 385-395 (2005).</li><li>Chen, X., Pham, E., Truong, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TEV+protease-facilitated+stoichiometric+delivery+of+multiple+genes+using+a+single+expression+vector.">TEV protease-facilitated stoichiometric delivery of multiple genes using a single expression vector.</a> Protein Sci. 19 (12), 2379-2388 (2010).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies.</a> J. Vis. Exp. (63), e3998 (2012).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. -. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J. Vis. Exp. (62), (2012).</li><li>Sukumaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+determination+of+nucleic+acids+and+proteins+using+the+micro-volume+BioSpec-nano-spectrophotometer.">Concentration determination of nucleic acids and proteins using the micro-volume BioSpec-nano-spectrophotometer.</a> J. Vis. Exp. (48), (2011).</li><li>JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Molecular Cloning. J. Vis. Exp Available from: <a target="_blank" href="https://www.jove.com/science-education/5074/molecular-cloning">https://www.jove.com/science-education/5074/molecular-cloning</a> (2016)</li><li>JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot. J. Vis. 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Co-ekspression af flere gener er i stigende grad afgørende, især ved karakterisering og rekonstituering af komplekse, multigen metabolske veje 3 , 4 , 5 . PMGX-systemet gør multigen co-ekspression i E. coli rutine 6 , 7 , 8 og tilgængelig for forskellige forskere. I denne undersøgelse viste fem proteiner af interesse, at ...

Samekspression af flere proteiner bliver stadig vigtigere, især i syntetiske biologiske applikationer, hvor flere funktionelle moduler skal udtrykkes 1 ; Ved undersøgelse af protein-proteinkomplekser, hvor udtryk og funktion ofte kræver coexpression 2 , 3 ; Og ved at karakterisere og udnytte biosyntetiske veje, hvor hvert gen i vejen skal udtrykkes 4 , 5 , 6 , 7 , 8 . Et antal systemer er blevet udviklet til co-ekspression, især i værtsorganismen Escherichia coli , arbejdshesten til rekombinant proteinekspression 9 . For eksempel kan multiple plasmider med forskellige selekterbare markører anvendes til at udtrykke individuelle proteiner ved anvendelse af et væld af forskellige ex<html> Pressionsvektorer 10 , 11 . Enkelte plasmidsystemer til multipel proteinekspression har anvendt enten multiple promotorer til at styre ekspressionen af ​​hvert gen 10 , 12 ; Syntetiske operoner, hvor flere gener kodes på et enkelt transkript 2 , 13 ; Eller i nogle tilfælde et enkelt gen kodende for et polypeptid, der i sidste ende behandles proteolytisk, hvilket giver de ønskede proteiner af interesse 14 . <img alt="figur 1" class="xfigimg" src="/files/ftp_upload/55187/55187fig1.jpg" /> Figur 1: pMGX-arbejdsgang viser konstruktionen af ​​en polykistronisk vektor. PMGX-systemet giver en fleksibel, nem at bruge strategi til konstruktion af syntetiske operoner under kontrol af en inducerbar T7-promotor.E.com/files/ftp_upload/55187/55187fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Venligst klik her for at se en større version af denne figur. I dette manuskript beskrives brugen af ​​et yderst effektivt system til konstruktion af multigen-syntetiske operoner under kontrol af en inducerbar T7-RNA-polymerase ( figur 1 ). Dette system tillader mange gener at udtrykkes samtidigt fra et plasmid. Det er baseret på et plasmidsystem, oprindeligt kaldet pKH22, der er blevet anvendt succesfuldt til en række forskellige applikationer 6 , 7 , 8 . Her ekspanderes dette plasmidsæt for at indbefatte fire beslægtede vektorer: pMGX-A, en ekspressionsvektor, der mangler nogen C- eller N-terminale mærker og med ampicillinresistensmarkøren; PMGX-hisA, en ekspressionsvektor, som koder for et N-terminalt hexahistidintag og med ampicillinresistensmarkøren; PMGX-K, en ekspressionsvektor lAkkingerer nogen C- eller N-terminale mærker og med kanamycinresistensmarkøren; Og pMGX-hisK, en ekspressionsvektor, som koder for et N-terminalt hexahistidintag og med kanamycinresistensmarkøren. I dette studie demonstreres fremgangsmåden til generering af en polykistronisk vektor indeholdende fem gener ved anvendelse af pMGX-systemet, specifikt pMGX-A, sammen med den succesrige produktion af hvert enkelt protein i Escherichia coli .

<table border="0" cellpadding="0" cellspacing="0" width="1224">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Enzymes</td>
			<td style="width:312px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alkaline Phosphatase, Calf Intestinal (CIP)</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">M0290S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AvrII</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0174S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EcoRI</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NdeI</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0111S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XbaI</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">R0145S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Herculase II Fusion DNA Polymerase</td>
			<td style="width:312px;">Agilent Technologies</td>
			<td style="width:139px;">600677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Name</td>
			<td style="width:312px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Reagents</td>
			<td style="width:312px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 kb DNA ladder</td>
			<td style="width:312px;">New England Biolabs</td>
			<td style="width:139px;">N3232L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-20% Mini-PROTEAN TGX Stain-Free Protein Gels</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">456-8095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 x TAE</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP1332-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP1423-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP160-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicilin</td>
			<td style="width:312px;">Sigma-Alrich</td>
			<td style="width:139px;">A9518-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BL21 (DE3) chemically comeptent cells</td>
			<td></td>
			<td style="width:139px;"></td>
			<td style="width:448px;">Comeptent cell prepared in house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B-PER Bacterial Protein Extraction Reagent</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td>PI78243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dNTP mix</td>
			<td style="width:312px;">Agilent Technologies</td>
			<td style="width:139px;"></td>
			<td>Supplied with polymerase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel Extraction Kit</td>
			<td style="width:312px;">Omega</td>
			<td style="width:139px;">D2500-02</td>
			<td>E.Z.N.A Gel Extraction, supplied by VWR Cat 3: CA101318-972</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP381-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">His Tag Antibody [HRP], mAb, Mouse</td>
			<td style="width:312px;">GenScript</td>
			<td style="width:139px;">A00612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immobilon Western Chemiluminescent HRP Substrate</td>
			<td style="width:312px;">EMD Millipore</td>
			<td style="width:139px;">WBKLS0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG</td>
			<td style="width:312px;">Sigma-Alrich</td>
			<td style="width:139px;">15502-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">BP1426-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">A411-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteurized instant skim milk powder</td>
			<td style="width:312px;">Local grocery store</td>
			<td style="width:139px;"></td>
			<td>No-name grocery store milk is adequate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Nitrocellulose membrane</td>
			<td style="width:312px;">Amersham Protran (GE Healthcare Life Sciences)</td>
			<td style="width:139px;">10600007</td>
			<td>Membrane PT 0.45 &#181;m 200 mm X 4 m, supplied by VWR Cat #: CA10061-086</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasmid DNA Isolation Kit</td>
			<td style="width:312px;">Omega</td>
			<td style="width:139px;">D6943-02</td>
			<td>E.Z.N.A Plasmid DNA MiniKit I, supplied by VWR Cat #: CA101318-898</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMGX</td>
			<td style="width:312px;">Boddy Lab</td>
			<td style="width:139px;"></td>
			<td>Request from the Boddy Lab Contact cboddy@uottawa.ca</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td style="width:312px;">Intergrated DNA Technologies</td>
			<td style="width:139px;"></td>
			<td>Design primers as needed for desired gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Synthetic Gene</td>
			<td style="width:312px;">Life Technologies</td>
			<td style="width:139px;"></td>
			<td>Design and optimize as needed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thick Blot Filter Paper</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">1703932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base</td>
			<td style="width:312px;">BioShop</td>
			<td style="width:139px;">TRS001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td style="width:312px;">Sigma-Alrich</td>
			<td style="width:139px;">P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XL1-Blue chemically competent cells</td>
			<td></td>
			<td style="width:139px;"></td>
			<td style="width:448px;">Comeptent cell prepared in house</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:327px;">Name</td>
			<td style="width:312px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Equipment</td>
			<td style="width:312px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioSpectrometer</td>
			<td style="width:312px;">Eppendorf</td>
			<td style="width:139px;">RK-83600-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel box - PAGE</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">1658005</td>
			<td>Mini-PROTEIN Tetra Vertical Electrophoresis Cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel Imager</td>
			<td style="width:312px;">Alpha Innotech</td>
			<td style="width:139px;"></td>
			<td>AlphaImager EC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator-oven</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">11-690-650D</td>
			<td>Isotemp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator-shaker</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">SHKE6000-7</td>
			<td>MaxQ 6000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Personna Razors</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">S04615</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Power Pack</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">S65533Q</td>
			<td>FB300</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transilluminator</td>
			<td style="width:312px;">VWR International</td>
			<td style="width:139px;">M-10E,6W</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermocylcer</td>
			<td style="width:312px;">Eppendorf</td>
			<td>Z316091</td>
			<td>Mastercycler Personal, supplied by Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Face-Shield</td>
			<td style="width:312px;"></td>
			<td style="width:139px;">18-999-4542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td style="width:312px;">Fisher Thermo Scientific</td>
			<td style="width:139px;">15-460-2SQ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Western Transfer Apparatus</td>
			<td style="width:312px;">Bio-Rad</td>
			<td style="width:139px;">1703935</td>
			<td>Mini-Trans Blot Cell</td>
		</tr>
	</tbody>
</table>

inducible t7 rna polymerase-mediated multigene expression system, pmgx

Co-ekspression af multiple proteiner er i stigende grad afgørende for syntetisk biologi, undersøgelse af protein-proteinkomplekser og karakterisering og udnyttelse af biosyntetiske veje. I dette manuskript beskrives anvendelsen af ​​et yderst effektivt system til konstruktion af multigen-syntetiske operoner under kontrol af en inducerbar T7-RNA-polymerase. Dette system tillader mange gener at udtrykkes samtidigt fra et plasmid. Her er et sæt af fire beslægtede vektorer, pMGX-A, pMGX-hisA, pMGX-K og pMGX-hisK, med enten ampicillin eller kanamycinresistens selekterbar markør (A og K) og enten besidder eller mangler en N-terminale hexahistidin Tag (hans) er beskrevet. Detaljerede protokoller til konstruktionen af ​​syntetiske operoner under anvendelse af dette vektorsystem tilvejebringes sammen med de tilsvarende data, hvilket viser, at et pMGX-baseret system indeholdende fem gener let kan konstrueres og anvendes til fremstilling af alle fem kodede proteiner i Escherichia coli . Denne systEm og protokol gør det muligt for forskere rutinemæssigt at udtrykke komplekse multikomponentmoduler og veje i E. coli .

I dette forsøg var målet at co-udtrykke fem proteiner fra et enkelt plasmid. De fem-kodonoptimerede syntetiske genfragmenter, der koder for enten N- eller C-terminale hexahistidinmærker, blev købt kommercielt. De syntetiske gener blev amplificeret ved PCR og individuelt klonet i en PCR-stump vektor og sekventeret. For at generere det polykistroniske plasmid klonedes de fem interesserede gener først i et egnet pMGX plasmid, pMGX-A. Figur 2 viser PCRBlunt...

Watch this Scientific Journal Video about Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX at JoVE.com

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

Denne undersøgelse beskriver metoder til T7-medieret co-ekspression af multiple gener fra et enkelt plasmid i Escherichia coli ved anvendelse af pMGX-plasmidsystemet.

Inducerbart T7 RNA Polymerase-medieret Multigen Expressions System, pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

The overall goal of this procedure is to construct multigene synthetic operons under the control of an inducible T7 RNA polymerase for expressing multiple proteins simultaneously. This method can help researchers in synthetic biology and biochemistry, enabling them to build multi-protein pathways or study protein complexes that are dependent on co-expression multiple approaches. The main advantage of this technique is that it allows multiple genes to expressed simultaneously in Escherichia coli from a single plasma.Demonstrating the procedure from my lab, will be Mohamed Hassan, a PhD student and Angela Thompson, a Co-op student. After obtaining and amplifying genes of interest according to the text protocol, prepare digestion reactions for the first gene of interest and the pMGX factor. Using a 40 microliter reaction containing 0.5 to 1.5 micrograms of DNA into the indel enzyme.Incubate the reactions at 37 degree Celsius for one hour. Then add EcoRI and allow the digest to proceed for an additional hour. NdeI is added prior to EcoRI to ensure that it digests effectively.NdeI is sensitive to the length in double-stranded DNA on either side of the restriction site. Adding it first ensures that it cuts the full-length circular plasma. Load the digestion reactions on to a 0.7%agarose gel and electrophores the samples at 100 volts for 55 minutes.Then, with a clean scalpel or razor blade, excise the insert and vector bands and place the excise gel segments into 1.5 milliliter tubes. Use a gel extraction kit according to the text protocol to purify the DNA. Set up a 20 microliter ligation reaction containing 0.15 to 0.5 micrograms of vector DNA and appropriate amount of insert depending on the size of the gene and one microliter of T4 DNA ligase.Under aseptic conditions, thaw on ice 100 microliter aliquots of chemically competent XL1-Blue E.coli cells for five minutes. And then add five microliters of the ligation reactions containing a gene of interest to the cells. Incubate the transformations on ice for 30 minutes.Next, heat shock the cells for 45 seconds in a 42 degree Celsius water bath. Then, place the transformations on ice and then add 200 microliters of cold LB medium. Leave the cells to incubate for two minutes.Incubate the cells at 37 degree Celsius in 220 rotations per minute for one hour and spread 100 microliters onto LB agar plates containing an appropriate selectable marker. To screen colonies for possible transformants, compare the number of colonies on the negative control versus the ligation plates. A colony count ratio of one to two is desired.Select four to eight individual colonies from each ligation reaction to inoculate 4 milliliters of LB with the appropriate antibiotic. Incubate the cultures at 37 degree Celsius and 220 rotations per minute overnight. With the plasmid DNA isolation kit, purify the plasmid DNA according to the text protocol and set up 20 microliter digestion reactions with 150 to 500 nanograms of DNA using one microliter each of NdeI and EcoRI.Incubate the digestion reactions at 37 degrees Celsius for two hours. To insert gene two into the pMGX vector containing gene one, setup a 40-microliter digestion reaction containing 0.5 to 1.5 micrograms of vector DNA containing the first gene of interest and one microliter of AVR2. Incubate the reaction at 37 degrees Celsius for 1.5 hours before adding 1.5 microliters of Calf-intestinal phosphatase.Incubate for an additional 30 minutes. Next, set up a 40 microliter digestion reaction containing 0.5 to 1.5 micrograms of the pMGX vector containing the second gene of interest. Using one microliter of AVR2 and xpol.Incubate the reaction at 37 degree Celsius for two hours. Electrophoris the restriction digests on 0.7 agarose gel. And use a clean scalpel eraser to excise the insert and vector bands as demonstrated earlier.After extracting the plasmid DNA and quantifying the sample according to the text protocol, set up a ligation reaction using a 3:1 insert to vector ratio of gene 2 and pMGX-yfg1. Include a negative control using pMGX-yfg1 only. Transform five microliters of each reaction into XL1-Blue cells as demonstrated earlier.Then compare the colony count on the negative control and ligation plates. If there are a large number of colonies on the negative control plate, review the CIP treatment. Select four to eight individual colonies from the ligation reaction and use each colony to inoculate four microliter of LB plus the appropriate antibiotic.Grow at 37 degrees Celsius and 220 rotations per minute overnight. After isolating and quantifying the plasmid DNA as before, screen for effective insertion of the second gene by setting up a digestion reaction containing 150 to 500 nanograms of DNA and one microliter of EcoRI. Incubate the reactions at 37 degrees Celsius for two hours.EcoRI is recommended for screening purposes but in some cases, EcoRI may generate two or more inserts that are similar in size which are difficult to distinguish on a gel. To ensure success, alternative restriction digest enzyme such as NdeI should be used. Separate the digestion reactions on an agarose gel, looking for a band that corresponds to the size of the inserted gene 2.Clone additional genes into the plasmid according to the text protocol. To generate the polycistronic plasmid, the five genes were first cloned into a suitable plasmid, pMGX A.This figure shows pCR-Blunt-yfg1 and pCR-Blunt-yfg2 in the pMGX A plasmid as well as pMGX A digested with indel and EcoRI. To clone the first two genes into pMGX A, the plasmid was digested with indel and EcoRI.After cloning, pMGX-yfg1 and pMGX-yfg2 where digested with AVR2 and xpol to confirm that successful clones were obtained. This figure shows the pMGXyfg1 vector digested with AVR2 and the pMGX-yfg2 vector digested with AVR2 and xpol to isolate gene of interest two. The resulting insert was cloned pMGX-yfg1 to create the pMGX-yfg1 two vector.As seen here, digestion of pMGX-yfg1 two with EcoRI confirmed the successful cloning of gene interest number two into pMGX-yfg1. This gel image demonstrates the difference between digestive plasmids that either had the insert in the correct orientation or in a reverse orientation. An insert in the reverse orientation would two EcoRI sites in close proximity making the resulting digested 50 base pair fragments undetectable on the gel and the backbone larger.Finally, after generating a five-gene polycistronic expression vector, a western blot of the expressed proteins confirms that all five proteins were expressed from a single vector. Note that the protein produced by yfg5 is the same size as yfg1 and is not separated on the gel. Once mastered, this technique can be completed in three or four days for every gene inserted into pMGX backbone if it is done correctly.While attempting this procedure, it's important to remember to treat and linearize vector with the phosphatase such as Calf-intestinal phosphatase to dephosphorylate the five vector. Following this procedure, further experiments such as in vivo multigene expression can be performed in order to characterize and harness biosynthetic pathways for the production natural products or to study protein-protein complexes. This technique paved the way for researchers in the synthetic biology field to produce complex carbohydrates and genetically engineered E.Coli.After watching this video, you should have a good understanding of how to implement the multigene expressions system in E.coli.

Research

JoVE Journal

Genetics

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Kinetik af efterslæb-DNA Synthesis<em&gt; In vitro</em&gt; Af bakteriofagen T7 replikationsproteiner

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Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

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